ProjectSummary MuchofthebasicbiologyofmRNAeditingremainsunknowndespitedecadesofstudy,largelydueto thelimitationsofexistingexperimentalmethods.AdenosinetoInosine(AtoI)editingaresultofadenosine deaminationcatalyzedbydeaminasessuchasADARenzymesisoneofthemostcommonformsof posttranscriptionalchemicalmodificationofRNA,generallyknownasRNAediting.AtoIeditingisessential fornotonlynoncodingRNAfunction,suchasrRNAandtRNAhigherorderstructurestabilization,butalsofor posttranscriptionalmRNAregulation.Forexample,GRIA2,whichencodestheGluR2subunitoftheAMPA glutamatereceptor,hasahighlyconservedADAR2targetededitingsiteinitscodingsequence.Perturbationof theorthologousratGria2editingeventleadstoneurologicaldysfunction.InhumanswithAmyotrophicLateral Sclerosis(ALS),manydiseaseaffectedmotorneuronsappeartodieasaresultofglutamatergiccalcium toxicityandhavedeficientGRIA2editing,andAdar2knockoutmicedemonstrateanALSlikephenotype,which isrescueduponexogenousexpressionofeditedGria2.GRIA2andseveralothermRNAAtoIeditingtargets havebeenstudiedformorethantwentyyears,andoverthelastfouryearstranscriptomewidesurveysofAto IeditinghaveprovidedevidenceformanymoremRNAtargets.Despitethismotivation,muchbasicinformation aboutthebiologyofthisimportantmRNAregulatoryprocess,includingitssubcellularlocalization,timing relativetotranscription,andassociationwithotherregulatoryfactors,remainsunknownlargelyduetothelimits ofexperimentaltechniques. Recently,ourlaboratorydescribedafluorescence?insitu?hybridizationmethodforvisualizingand quantifyingsinglenucleotidevariants(SNVs)insinglecellsusinganovelprobehybridizationstrategyknown asSNVFISH.Weareleveraginginosine'sstructureasaguanosineanalogtoadaptSNVFISHtotheanalysis ofAtoIeditinginaprotocolwerefertoasinosineFISH(iFISH).Ourpreliminarydatashowthatwecanuse iFISHtospecificallyidentifyeditedanduneditedGRIA2transcriptsi?nsitu?inculturedcells.Inthisproject,we arequantifyingsubsinglecellulartrendsinAtoIeditingofknowntargets,suchasGRIA2andmRNAtargets newlyidentifiedintranscriptomewidescreens.Specifically,weareusingiFISHtomeasuresinglecellediting ratedistributions,tovisualizesubcellularlocalizationpatternsofeditedmRNAs,andtomeasureeditingtiming relativetotranscription.WearealsostudyingtheeffectofADAR2onsubcellularGRIA2trafficking.Lastly,we areapplyingournovelmethodtowardbettercharacterizingperturbationsofGRIA2editinginindividualmotor neuronsinALSlesionsbyextendingtheuseofiFISHtofixedsectionsofpostmortembrainandspinalcord tissuesamplesfromhealthyanddiseaseddonors.